Photodynamic therapy (PDT), a cancer treatment utilizing a photosensitizer, visible light, and oxygen, is a unique oxidative stress that generates singlet oxygen, extranuclear cytotoxic damage, and a rapid induction of apoptosis in vitro and in vivo. PDT also produces ceramide, a lipid second messenger that is associated with stress responses and induces apoptosis. The proposes research will test the hypothesis that ceramide mediates apoptosis in response to PDT. A431 human epidermoid carcinoma cells, that respond to PDT by an increased ceramide accumulation and apoptosis, will be studied, as well as EBV-transformed normal or Niemann-Pick human lymphoblasts, the latter lacking acidic sphingomyelinase (SMase). The photosensitizers to be used, the phthalocyanine Pc 4 and merocyanine-540, differ in subcellular localization and in their ability to causes apoptosis. The hypothesis will be tested in four Specific Aims. For Aim 1, ceramide and sphingomyelin levels, SMase activities, cell survival, and apoptosis will be measured to define the response to PDT and to assess the importance of survival, and apoptosis will be measured to define the response to PDT and to assess the importance of acidic SMase deficiency. In order to determine if an increase in reactive oxygen species is linked to PDT-induced apoptosis via ceramide, for Aim 2, the cellular and mitochondrial levels of superoxide anion and ceramide will be measured after PDT, as well as the influence of superoxide dismutase mimics and the anti-apoptotic oncoprotein Bcl-2 on ceramide generation and apoptosis. In Aim 3, the activity of stress- activated protein kinases will be determined and the roles of c-Jun, apoptosis-associated caspases, and Bcl-2 will be assessed with respect to their coupling to ceramide-mediated PDT-induced apoptosis. In Aim 4, the influence of tumor necrosis factor as a mediator in ceramide and apoptotic responses to PDT will be evaluated. The proposed studies should elucidate mechanisms of apoptosis initiating in specific membrane sites, identify early biochemical responses driven by PDT-associated ceramide and suggest methods to improve the therapeutic efficacy of PDT.